Modulation of deubiquitinase family members

ABSTRACT

Modulation of Deubiquitinase Family Members The present invention relates to the therapeutic treatment of conditions by modulating members of the deubiquitinase family, and also to assays for identifying substances which may be useful in such treatments, as well as to the treatment of cylindromatosis and more generally to the modulation of other conditions associated with activation of the transcription factor NF-κB, such as inflammation.

FIELD OF THE INVENTION

The present invention relates to the therapeutic treatment of conditionsby modulating members of the deubiquitinase family, and also to assaysfor identifying substances which may be useful in such treatments.

In one aspect, the invention relates generally to assay methods foridentifying modulators of HIF-α, where the assay involves identifyingsubstances which bind to and/or modulate an activity of VDU1. It alsorelates to modulators of VDU1 for use in methods of medical treatment,and in particular, in the treatment of conditions which can be improvedby modulating the activity of HIF.

In another aspect, the invention relates to the treatment ofcylindromatosis and more generally to the modulation of other conditionsassociated with activation of the transcription factor NF-κB, such asinflammation.

BACKGROUND TO THE INVENTION

HIF

The transcription factor HIF (hypoxia inducible factor) system is a keyregulator of responses to hypoxia, occupying a central position inoxygen homeostasis in a wide range of organisms. A large number oftranscriptional targets have been identified, with critical roles inangiogenesis, erythropoiesis, energy metabolism, inflammation, vasomotorfunction, and apoptotic/proliferative responses. The system is essentialfor normal development, and plays a key role in pathophysiologicalresponses to ischaemia/hypoxia. HIF is also important in cancer, inwhich it is commonly up-regulated, and has major effects on tumourgrowth and angiogenesis.

The HIF DNA binding complex consists of a heterodimer of α and βsubunits. Regulation by oxygen occurs through the α-subunits, which arerapidly destroyed by the proteasome in oxygenated cells. This involvestargeting of HIF-α subunits by the von Hippel-Lindau tumour suppressor(pVHL), with pVHL acting as the recognition component of a ubiquitinligase that promotes ubiquitin dependent proteolysis through interactionwith a specific sequence in HIF-α-subunits. In hypoxia, this process issuppressed, so stabilizing HIF-α and permitting transcriptionalactivation.

CYLD

Cylindromas are rare benign adnexal tumours that arise primarily on thescalp in humans. They occur at any age but usually appear in earlyadulthood. There are 2 distinct clinical forms: a solitary form, whichis sporadic, and a multiple form, which is dominantly inherited,referred to as familial cylindromatosis. The lesions are pink-red,nodular, firm, and usually painless, and vary in size from severalmillimetres to more than 6 cm in diameter. The tumours grow slowly insize and number throughout life; in severe cases, they may cover theentire scalp and are known as “turban tumours.” At present, treatment isby surgery to remove the tumours followed by reconstruction of theaffected area.

The condition has been linked to the chromosomal region 16q12-13 andmore recently Bignell, G. R. et al., (Nat Genet 25, 160-5 (2000))identified the gene, CYLD, in this region which is mutated inindividuals with cylindromatosis. The gene is regarded as a tumoursuppressor gene though its mode of action has not been elucidated.

Nuclear factor κB (NF-κB) is a sequence-specific transcription factorthat is known to be involved in the inflammatory and innate immuneresponses. NF-κB is activated by release from an inhibitory factor,referred to as IkB. NF-κB is a heterodimer consisting of a 50 kDa (p50)and a 65 kDa (p65) DNA-binding subunit. NF-κB contributes to theso-called “immediate-early” activation of defence genes if cells areexposed to primary or secondary pathogenic stimuli.

It has also been found that NF-κB and the signalling pathways that areinvolved in its activation are also important for tumour development, inthat NF-κB also regulates cell proliferation and apoptosis. NF-κB hasbeen shown to be constitutively activated in several types of cancercell.

DISCLOSURE OF THE INVENTION

The present inventors have identified a regulatory pathway present incells in which HIF-α is stabilised by the action of the VHL-interactingdeubiquitinase enzyme 1 (VDU1).

Previously, no target for deubiquitination by VDU1 has been identifiedand so no physiological role has been demonstrated.

The activity of VDU1 represents a novel target for the control of HIF-α.Loss of VDU1 leads to a decrease in HIF-α, and a reduction in theresponses mediated by HIF. Accordingly, reduction of VDU1 may be usefulin the treatment of diseases associated with inappropriate HIF activity,or in other conditions in which reduction of HIF activity can have sometherapeutic benefit.

Conversely, it is believed that increasing VDU1 activity will stabiliseHIF-α and lead to an increase of HIF-mediated responses, which may bebeneficial for example in promoting new vascular growth.

The finding that HIF-α stability can be regulated by VDU1 provides for anovel assay method for the development of new agents for human or animaltherapy.

In one aspect, assays of the invention are generally directed atdetermining whether a test substance is capable of modulating thestability and/or state of ubiquitination of HIF-α, via modulation ofVDU1.

This may be done by:

bringing into contact a putative modulator and a VDU1 polypeptide;

determining whether the putative modulator binds and/or modulates anactivity of VDU1;

determining the effect of the putative modulator on HIF-α stabilityand/or on the ubiquitination state of HIF-α in a test system comprisingHIF-α and VDU1.

Accordingly, in one aspect, the invention provides an assay method whichincludes:

bringing into contact a VDU1 polypeptide with a putative modulator;

determining binding between the VDU1 polypeptide and the putativemodulator;

bringing the putative modulator into contact with a test systemcomprising VDU1 and HIF-α; and

determining the effect of the putative modulator on the stability and/orstate of ubiquitination of HIF-α.

It is known that VDU1 and HIF-α both bind to the β-domain of VHL, andthat VHL targets both these proteins for ubiquitination (Li et al,2002). In the light of the present finding that VDU1 stabilises HIF-α,it is believed that the binding of VDU1 to VHL may bring VDU1 intophysical proximity with HIF-α and so may facilitate the interactionbetween VDU1 and HIF-α.

Also, it is believed that an agent which blocks the ability of VDU1 tobind VHL may reduce the extent to which HIF-α is stabilised by VDU1,and/or may alter the state of ubiquitination of HIF-α, and hence reducethe activity of HIF in the cell.

Accordingly, in another aspect of the invention, there is provided anassay method which includes:

bringing into contact a VHL polypeptide, a VDU1 polypeptide and aputative modulator;

determining whether the putative modulator modulates the interaction ofthe VHL and VDU1 polypeptides;

bringing the putative modulator into contact with a test systemcomprising VDU1, VHL and HIF-α;

determining the effect of the putative modulator on the stability and/orstate of ubiquitination of HIF-α.

In this aspect, the relevant activity of VDU1 is its ability to bindVHL.

In a further aspect of the invention, there is provided an assay methodcomprising:

bringing a putative modulator into contact with VDU1 and anubiquitinated VDU1 substrate;

determining the ability of the putative modulator to modulate thestabilisation and/or state of ubiquitination of the substrate by VDU1;

bringing the putative modulator into contact with a test systemcomprising VDU1 and HIF-α;

determining the effect of the putative modulator on the stability and/orstate of ubiquitination of HIF-α.

In this aspect, the relevant activity of VDU1 is the ability todeubiquitinate and stabilise a substrate.

Determining whether a test substance is capable of modulating thestability and/or state of ubiquitination of HIF-α via modulation of VDU1can also be done, in further aspect of the invention, by:

bringing into contact a putative modulator with a test system comprisingVDU1 and ubiquitinated HIF-α;

determining the ability of the putative modulator to modulate thestabilisation and/or state of ubiquitination of HIF-α by VDU1.

In the assays above, the test system can optionally comprise VHL,especially when the assay is an assay for an inhibitor.

Specific modulators of VDU1 have not previously been shown to be usefulfor methods of therapy. Accordingly, the invention also provides amodulator of VDU1 for use in a method of medical treatment, and, inanother aspect, a composition comprising a modulator of VDU1 and apharmaceutically acceptable excipient.

In a further aspect, the invention provides the use of a modulator ofVDU1 for the manufacture of a medicament for the treatment of acondition in which modulation of HIF is of therapeutic value.

In a still further aspect, the invention provides a method of treating adisease in which modulation of HIF is of therapeutic value, the methodcomprising administering to an individual an effective amount of anagent which modulates the activity of VDU1.

The present inventors have also identified a regulatory pathway presentin cells which directly links the action of CYLD to the suppression ofNF-κB. Loss of CYLD thus leads to an increase in NF-κB activity, whichin turn causes an increase in anti-apoptopic gene function. This mayresult in the disruption of the balance of pro- and anti-apoptopic generegulation in cells of the skin, leading to the growth of the benigntumours associated with cylindromatosis.

Thus in another aspect, the invention provides a method of treating anindividual with cylindromatosis by administering to the individual aneffective amount of an NF-κB inhibitor.

In further aspect, the invention provides the use of an NF-κB inhibitorfor the manufacture of a medicament for the treatment ofcylindromatosis. Alternatively, the invention provides an NF-κBinhibitor for use in a method of treatment of cylindromatosis.

In another aspect, the finding that the action of CYLD is to suppressNF-κB activity provides a new target for the treatment of diseasesassociated with activation of NF-κB. Thus the invention provides amethod of treating such a disease in an individual by administering tothe individual an effective amount of an agent which increasesexpression of CYLD. In another aspect, the invention provides the use ofan agent which increases expression of CYLD for the manufacture of amedicament for the treatment of a disease associated with activation ofNF-κB. Alternatively, the invention provides an agent which increasesexpression of CYLD for use in a method of treatment of a diseaseassociated with activation of NF-κB.

In a further aspect, the finding that NF-κB activity can be regulated byCYLD provides a novel assay method for the development of new agents forhuman or animal therapy. Thus the invention provides an assay methodwhich includes the steps of:

providing a cell culture in which CYLD activity is suppressed ormissing;

bringing the culture into contact with an agent to be assayed; and

determining the effect of the agent on the activity of NF-κB.

These and other aspects of the invention are described further hereinbelow.

DESCRIPTION OF THE DRAWINGS

FIG. 1—Inverse values of the 3×RE-luciferase HIF-1α responsive reporterand individual members of the DUB knockdown library under conditionsthat mimic hypoxia in HEP-G2 cells (12 hrs 1 mM. Desferrioxamine (DFO)exposure starting 48 hrs after transfection). SV40 Renilla luciferaseserved as an internal control.

FIG. 2—Activity of the 3×RE luciferase reporter in the presence andabsence of pSUPER-VDU-1 under normoxic conditions and conditions thatmimic hypoxia (12 hrs DFO). SV40 Renilla luciferase served as aninternal control.

FIG. 3—U2-OS cells were transfected with pSUPER or pSUPER-VDU-1 andexposed to 1 mM DFO for 12 hrs. Whole cell extracts were immunoblottedwith a Hif1-α specific antibody.

FIG. 4 CYLD is an antagonist of NF-κB signalling. U2-OS cells weretransfected with a NF-κB luciferase reporter and pSUPER-CYLD or emptyvector. Forty-eight hours after transfection cells were stimulatedovernight with PMA (200 nM) or TNF-α (20 ng/ml) and luciferase activitywas measured. SV40 Renilla luciferase served as an internal control.

DETAILED DESCRIPTION OF THE INVENTION

HIF

The amino acid sequence of human VDU1 is given in Li et al (2002), andis also given in Genbank reference AF383172. At least two putativesubtypes are known. Type I consists of 942 amino acids and type IIconsists of 911 amino acids with predicted molecular masses of 107 and103 kDa, respectively. In the present application, VDU1 will beunderstood to be any suitable mammalian VDU1, preferably human VDU1,including alleles, homologs and orthologs of the known sequences.Although wild type sequences are preferred, the term VDU1 will also beunderstood to include variants, provided they retain the ability todeubiquitinate HIF-α. Preferably, the variants also retain the abilityto bind VHL.

Generally it is preferred that variants have a degree of amino acididentity which is desirably at least 70%, preferably at least 80%, 90%,95% or even 98% to a wild type mammalian VDU1, preferably to human VDU1.

VHL has also been cloned, and the sequence of human VHL is available asGenbank accession numbers AF010238 and L15409.

A number of HIF-α subunit proteins have been cloned. These includeHIF-1α, the sequence of which is available as Genbank accession numberU22431; HIF-2α, available as Genbank accession number U81984; andHIF-3α, available as Genbank accession numbers AC007193 and AC079154.These are all human HIF-α subunit proteins.

In the present application, VHL and HIF-α will be understood to be anysuitable mammalian VHL or HIF-α, preferably human, including alleles,homologs and orthologs of the known sequences. HIF-α is preferablyHIF-1α.

Although wild type proteins are preferred, references to the HIF-α andVHL in these assay methods also include reference to variants andfragments which retain a relevant function of the wild type protein. Inthe assay methods discussed herein, suitable VHL variants are thosewhich retain the ability to bind HIF-α and VDU1. More preferably, theyalso retain the ability to target HIF-α for ubiquitination. SuitableHIF-α variants preferably retain the ability to be labelled withubiquitin, and to be recognised for de-ubiquitination by VDU1. Morepreferably, they also retain the ability to bind VHL. In someembodiments, they retain the ability to bind to and/or activate aresponse element.

Generally it is preferred that variants have a degree of amino acididentity which is desirably at least 70%, preferably at least 80%, 90%,95% or even 98% to a wild type mammalian HIF-A or VHL, preferably tohuman HIF-α or VHL.

Sequence identity can be assessed using the algorithm BLAST 2 SEQUENCES,using default parameters.

Assay Methods

Assay methods of the invention in this aspect provide modulators of VDU1activity which are useful in treating conditions in which HIF activityis harmful or may be beneficial. These conditions are discussed in moredetail below.

In some aspects of the invention, the assay methods involve a firststage of assessing whether the putative modulator binds to a VDU1polypeptide, or affects the interaction between VDU1 and VHLpolypeptides.

In respect of the first stage of these assay methods, it will beunderstood that the term “VHL polypeptide” or “VDU1 polypeptide”includes reference to VDU1 and VHL as defined above, but also includesfragments of these proteins. Generally fragments, where used, will be atleast 40, preferably at least 50, 60, 70, 80 or 100 amino acids in size.Where the assay involves the assessment of binding between VDU1 and VHL,then fragments of any size may be used, provided that they retain theability to bind to each other in the absence of a test compound.

Preferred VHL fragments for use in assessing the VDU1/VHL interactioninclude those which are based at least in part upon the beta domainlocated within the fragment 63-156 of the 213 amino acid human VHLprotein, or the equivalent domain in other variants. In a preferredembodiment, such domains will have at least 70%, preferably 80%, 90%,95% or even 98% degree of sequence identity to the 64-156 fragment ofhuman VHL. Fragments of this region and its variants may be used. Thesefragments may be 15-80 amino acids in length, for example from 20 to 80,such as 30-60 amino acids in length. Desirably, the wild-type sequenceof the beta domain is retained.

Fragments may include the region 63-83 of human VHL or their equivalentsin the above described variants.

One fragment which may be used is that in which up to 53 of theN-terminal residues, e.g. from 1 to n wherein n is an integer of from 2to 53, have been deleted, the rest of the protein being wild-type.

Fragments may be generated and used in any suitable way known to thoseof skill in the art. Suitable ways of generating fragments include, butare not limited to, recombinant expression of a fragment from encodingDNA. Such fragments may be generated by taking encoding DNA, identifyingsuitable restriction enzyme recognition sites either side of the portionto be expressed, and cutting out said portion from the DNA. The portionmay then be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments (e.g. up to about 20 or 30 amino acids) may also begenerated using peptide synthesis methods which are well known in theart.

Where the assay method includes bringing into contact a VHL polypeptide,a VDU1 polypeptide and a putative modulator, and determining whether theputative modulator modulates the interaction of the VDU1 and VHLpolypeptides (i.e., in the second aspect of the invention describedabove), it will also be understood that the terms “VDU1 polypeptide” and“VHL polypeptide” are also intended to refer to variants of VHL or VDU1other than those described above, provided they retain the ability tobind VDU1 or VHL, respectively.

The step of bringing into contact a VHL polypeptide, a VDU1 polypeptideand a putative modulator compound may be done under conditions where theVHL polypeptide and the VDU1 polypeptide, in the absence of modulator,are capable of forming a complex.

In an alternative embodiment, the step may be carried out in conditionswhere the association does not occur in the absence of the modulator.This may be desirable for looking for agents which enhance or potentiatethe binding.

Determining the effect of the putative modulator on the binding of VHLand VDU1 may be done in the presence and absence of the modulator. Achange, i.e. an increase or decrease in binding in the presence relativeto the absence of the putative modulator will be indicative of theability of the putative modulator to modulate the interaction.

Generally, the method of determining binding or modulation of complexformation is not part of the present invention and the skilled personmay use any of the methods known in the art.

To identify the binding of small molecules to polypeptides, standardassay formats may be used. For example, the polypeptide may beimmobilised on a support, and a known amount of small molecule or adetectably labelled small molecule may be added to the protein. Theinteraction may be measured, for example, as described below in relationto in vitro assays for protein-protein interaction.

One assay format which is widely used in the art to study theinteraction of two proteins is a two-hybrid assay. This assay may beadapted for use in the present invention. A two-hybrid assay comprisesthe expression in a host cell of the two proteins, one being a fusionprotein comprising a DNA binding domain (DBD), such as the yeast GAL4binding domain, and the other being a fusion protein comprising anactivation domain, such as that from GAL4 or VP16. In such a case thehost cell (which again may be bacterial, yeast, insect or mammalian,particularly yeast or mammalian) will carry a reporter gene constructwith a promoter comprising DNA binding elements compatible with the DBD.The reporter gene may be a reporter gene such as chloramphenicol acetyltransferase, luciferase, green fluorescent protein (GFP) andβ-galactosidase, with luciferase being particularly preferred.

Two-hybrid assays may be in accordance with those disclosed by Fieldsand Song, 1989, Nature 340; 245-246. In such an assay the DNA bindingdomain (DBD) and the transcriptional activation domain (TAD) of theyeast GAL4 transcription factor are fused to the first and secondmolecules respectively whose interaction is to be investigated. Afunctional GAL4 transcription factor is restored only when two moleculesof interest interact. Thus, interaction of the molecules may be measuredby the use of a reporter gene operably linked to a GAL4 DNA binding sitewhich is capable of activating transcription of said reporter gene.

Thus two hybrid assays may be performed in the presence of a potentialmodulator compound and the effect of the modulator will be reflected inthe change in transcription level of the reporter gene constructcompared to the transcription level in the absence of a modulator.

Host cells in which the two-hybrid assay may be conducted includemammalian, insect and yeast cells.

The interaction of VHL and VDU1 may also be examined directly, forexample using microcalorimetry.

Another assay format measures directly the interaction between VDU1 andVHL by labelling one of these proteins with a detectable label andbringing it into contact with the other protein which has beenoptionally immobilised on a solid support, either prior to or afterproteins have been brought into contact with each other. Suitabledetectable labels include ³⁵S-methionine which may be incorporated intorecombinantly produced proteins, and tags such as an HA tag, GST orhistidine. The recombinantly produced protein may also be expressed as afusion protein containing an epitope which can be labelled with anantibody. Alternatively, an antibody against VDU1/VHL can be obtainedusing conventional methodology.

The protein which is optionally immobilized on a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se.

Alternatively, the interaction of the proteins may be measured byimmunoprecipitation of one followed by immunological detection of theother, e.g. by western blotting or electrophoretic mobility ofdetectably labelled proteins.

In a further alternative mode, one of VDU1 and VHL may be labelled witha fluorescent donor moiety and the other labelled with an acceptor whichis capable of reducing the emission from the donor. This allows an assayaccording to the invention to be conducted by fluorescence resonanceenergy transfer (FRET). In this mode, the fluorescence signal of thedonor will be altered when VDU1 and VHL interact. The presence of acandidate modulator compound which modulates the interaction willincrease or decrease the amount of unaltered fluorescence signal of thedonor.

FRET is a technique known per se in the art and thus the precise donorand acceptor molecules and the means by which they are linked to VDU1and VHL may be accomplished by reference to the literature.

Suitable fluorescent donor moieties are those capable of transferringfluorogenic energy to another fluorogenic molecule or part of a compoundand include, but are not limited to, coumarins and related dyes such asfluoresceins, and suitable acceptors include, but are not limited to,coumarins and related fluorophores, and the like.

Another technique which may be used is a scintillation proximity assay(reagents and instructions available from Amersham Pharmacia Biotech) inwhich a target compound (i.e. for this invention VHL, VDU1) is held on(or in the course of the assay attached to) a bead having a signallingcompound which scintillates when activated by radioactivity emitted by aradiolabel attached to a target-binding molecule (i.e. for thisinvention another of VHL or VDU1).

In another aspect of the invention, the assay comprises the first stepof bringing a putative modulator into contact with VDU1 and anubiquitinated VDU1 substrate, and determining the ability of theputative modulator to modulate the stabilisation and/or state ofubiquitination of the substrate by VDU1.

An “ubiquitinated substrate” herein refers to a molecule conjugated toone or more ubiquitin moieties, which is a substrate fordeubiquitination, e.g., by VDU1.

Ubiquitinated VDU1 substrates which may be used in the above methodsinclude for example ubiquitinated GST or ubiquitinatedbeta-galactosidase.

This first step is preferably carried out in vitro. In this embodiment,the ubiquitinated substrate may be labelled, e.g, with [³⁵S] methionine.

The ubiquitinated substrate may in some embodiments be provided by thepresence of an active ubiquitination system, but in other embodiments,ubiquitinated substrate may be provided, e.g., by isolation of thesubstrate from a cell or by in vitro ubiquitination.

Alternatively, the first step of this assay may be carried out in a cellexpressing the substrate (optionally a labelled substrate), preferably amammalian cell line, more preferably a human cell line.

Methods of determining the ability of a putative modulator to modulatethe stabilisation of a substrate by VDU1 are discussed below, inrelation to HIF-α. Unless otherwise apparent from the context, thesemethods also apply to other VDU1 substrates. Certain preferred formatsfor determining the ability of the modulator to modulate thestabilisation of the substrate will be apparent from this discussion.

In the aspects of the invention discussed above, the assay method alsoincludes a step of contacting a test system comprising HIF-α and VDU1with the modulator, and determining the effect on HIF-α stability and/oron the ubiquitination state of HIF-α. In some embodiments, VHL is alsopresent in the test system.

The test system could be an in vitro test system. In one embodiment, thetest system may comprise labelled HIF-α, e.g., labelled with[³⁵S]methionine. The test system may also comprise cell extract, whichmay be the source of the VDU1, HIF-α and/or VHL. The cell extract can beobtained from any cell, and is preferably obtained from one of the celllines described below.

In order to assess the effect of the deubiquitinase, the HIF-α in thetest system is preferably conjugated to one or more ubiquitin moieties,at least transiently. This may in some embodiments be achieved by thepresence of VHL and other components of the ubiquitination system,together with free ubiquitin. In other embodiments, ubiquitinated HIF-αmay be provided, e.g., by isolation of HIF-α from a cell, especially anormoxic cell, or by in vitro ubiquitination. In vitro ubiquitination ofHIF-α may be achieved using a reconstituted complex of VHL, Rbx1, Cul2,Elongin B and Elongin C (see for example Kamura et al 2000, PNAS vol.97, no. 19: 10430-10435).

In another embodiment, the test system could be a cell. Assays accordingto the invention may be performed in any cell line expressing HIF-α,preferably one in which the HIF-α ubiquitination system is active,preferably a mammalian cell line, more preferably a human cell line.

In some embodiments, the cell line may express a labelled version ofHIF-α, e.g., labelled with a histidine tag, to allow isolation of theprotein.

In some embodiments of the invention, the cell may be under hypoxicconditions. Under these conditions the HIF pathway will be at a highlevel of activation. This may be preferred for example when themodulator is an inhibitor of VDU1, and so decreases the stability ofHIF-α.

In other embodiments of the invention, the cell may be under normoxicconditions. Under normoxic conditions, the HIF pathway will generally beat a low level of activation. This may preferred for example when themodulator is an activator of VDU1, and so will increase the stability ofHIF-α.

The stability of HIF-α in an assay method of the invention may bedetermined by a variety of means. For example, where the effect of thetest compound on HIF activity is assessed in vitro, the effect may beassessed by determining the level of ubiquitination of HIF-α, forexample by determining the change in molecular weight or by isolatingHIF-α (e.g., by immunoprecipitation) and then immunoblotting withantibodies against ubiquitin. Where the effect of the test compound isassessed in a cell, it is also possible to assess the amount of HIF-α inthe cell, e.g., using Western blotting. Additionally, the activity ofHIF-α can be examined using a reporter gene assay (e.g., fireflyluciferase, secreted alkaline phosphatase or green fluorescent protein)whose promoter comprises a target site recognised by HIF, e.g., apromoter from the VEGF or erythropoietin genes.

It is preferred that determining the effect of the modulator on thestability of HIF-α will be carried out under conditions where VDU1 iscapable of stabilising HIF-α in the absence of the modulator. This isparticularly preferred where the assay is for inhibitors of VDU1activity.

In an alternative, the assay may be carried out under conditions whereVDU1 cannot stabilise HIF-α in the absence of the modulator, which maybe desirable for example when the assay is an assay for an activator.

Determining the effect of the putative modulator on the stability ofHIF-α may be done in the presence and absence of the modulator. Achange, i.e. an increase or decrease in HIF-α stability in the presencerelative to the absence of the putative modulator will be indicative ofthe ability of the putative modulator to modulate HIF-α stability.

Because the assays above comprise a preliminary step of assessingbinding to VDU1 or modulation of an activity of VDU1, then it will beapparent that, e.g., changes in reporter gene expression are due to achange in the action of VDU1 on HIF-α and hence represent a change instability. However, it may be preferred that stability is measureddirectly, e.g., by measuring a change in the amount of protein or morepreferably by measuring a change in ubiquitination state of HIF-α, asbelow.

In an alternative aspect of the invention, the assay method includes:

bringing into contact a putative modulator with a test system comprisingVDU1 and ubiquitinated HIF-α;

determining the ability of the putative modulator to modulate thestabilization and/or state of ubiquitination of HIF-α by VDU1.

The test system may be a test system as described above, althoughcertain preferred embodiments will be apparent from the followingdiscussion.

In this assay, it is necessary to determine the ability of the modulatorto modulate the stabilisation of HIFα by VDU1, rather than, for example,the ability to directly affect HIF-α, or to affect the ubiquitinationpathway. This can be done by eliminating the other possibilities, invarious ways as will be apparent to the skilled person in the light ofthe present disclosure.

The following discussion applies also to determining the ability of aputative modulator to modulate the stabilisation of other ubiquitinatedVDU1 substrates, as discussed above.

For example, in order to eliminate the possibility that the modulator isacting on the ubiquitination pathway rather than the deubiquitinationpathway, the assay may be carried out under conditions where theubiquitination pathway is not active. This may be achieved by carryingout the assay in the absence of a factor, e.g., a protein, which isrequired for substrate ubiquitination. The absence may be a totalabsence from the system, or may be absence in a functional form. Forexample, where the ubiquitinated substrate is HIF-α, the assay may becarried out in the absence of a component of the E3 ubiquitin ligase,such as elongin C, elongin B, cullin-2 or rbx-1. In some embodiments,the assay may be carried out in the absence of VHL.

Where no ubiquitination activity is present, the substrate will need tobe provided in an ubiquitinated form. This may be done for example by invitro ubiquitination, or by isolating the substrate from a cell. In thecase of HIF-α, this is especially a cell under normoxic conditions. Themost convenient format for such an assay will be in vitro. The testsystem may comprise cell extract in some embodiments, e.g., an extractfrom a cell deficient in a relevant ubiquitinase activity (such as aHIF-α ubiquitinase activity).

Methods of assessing HIF-α stability are described above, and thesemethods may also be used to determine the ability of the putativemodulator to modulate the stabilisation of HIF-α (or, where applicable,other ubiquitinated VDU1 substrate) by VDU1.

In order to exclude the possibility that the modulator interactsdirectly with HIF-α (or, where applicable, other substrates), the assaypreferably involves directly assessing the ubiquitination state of thesubstrate, e.g., by detecting a change in molecular weight or byimmunoblotting with antibodies against ubiquitin.

Another method of confirming that the test substance is able to modulatethe stabilisation of HIF-α (or other substrates) by VDU1 is to carry outcontrol experiments, which the skilled person will be able to design inthe light of the present disclosure using his general skill andknowledge. For example, in order to confirm that a test substance ismodulating deubiquitination and not, for example, modulating theubiquitination pathway, it would be possible for the skilled person totake a different test system, comprising the ubiquitination system and asubstrate thereof (but without a functional deubiquitination pathway),and determine whether the putative modulator can modulate theubiquitination state of the substrate in that system. Similarly, toconfirm that the effect is not due to a direct modulation of HIF-α, itwould be possible to take a test system comprising HIF-α and a reportergene, and to determine whether the modulator has any effect in this testsystem. Other control experiments will be apparent to the skilledperson.

In each of the assay methods of the invention described above, theamount of agent which may be used will normally be determined by trialand error. Typically, from about 1 nm to 100 μm concentrations of agentcompounds may be used, for example from 0.1-10 μm. Agent compounds whichmay be used may be natural or synthetic compounds used in drug screeningprograms. Extracts of plants or microorganisms which contain severalcharacterised or uncharacterised components may also be used.

Therapeutic Methods and Uses.

As indicated above, HIF is a transcription factor having a number ofknown transcriptional targets. There are therefore a number of diseasesin which reduction or enhancement of HIF activity may be of value intreatment.

Diseases in which reduction of HIF activity may be of value may be thoseassociated with HIF activity, more preferably with HIF1 activity.Preferably, they are diseases which are associated with inappropriateangiogenesis or with inflammation. Specific examples include cancer (seefor example Cramer et al, 2003), eye diseases such as maculardegeneration and diabetic retinopathy (Witmer et al, 2003), Alzheimer's(Vagnucci et al, 2003), atherosclerosis (Ross J S et al, 2001),psoriasis (Dredge et al, 2002), rheumatoid arthritis (Dredge et al,2002) endometriosis (Healy et al, 1998), and the like.

Enhancement of HIF activity may be useful for example when new vasculargrowth and/or promotion of cellular survival or cellular function inhypoxia is of benefit, e.g., in peripheral or coronary artery diseases(Kusumanto et al, 2003) or in myocardial ischaemia and the like.Additionally, vasomotor control can be regulated by HIF, and soactivation of HIF might lower systemic blood pressure.

By “treatment”, is meant any degree of alleviation of the disease,including slowing its development. This will be beneficial in increasingthe time until alternative treatment (such as surgery) is required.Treatment is also intended to include prophylaxis, e.g., to preventischaemia, for example in the promotion of coronary collaterals in thetreatment of angina.

In the present invention these diseases may be treated by theadministration of a modulator of VDU1.

By “modulator” of a protein (e.g., VDU1), is meant an inhibitor oractivator, i.e., an agent which reduces or enhances the total activityof the protein.

By “inhibitor” is meant an agent which reduces the total activity of theprotein. This may be by reducing the total amount of protein in thecell, preferably by reducing the expression of that protein.Alternatively, it may be inhibition which occurs by reducing the abilityof a protein to perform its function.

Similarly, by “activator” is meant an agent which increases totalprotein activity. This may be by increasing the total amount of proteinin the cell, preferably by enhancing expression, or by increasing theability of the protein to perform its function.

The terms “modulation”, “inhibition” and “activation” are to beinterpreted accordingly.

Preferably, a modulator of VDU1 is a specific modulator, that is, onewhich directly affects the protein activity of VDU1 but does notdirectly affect the stability of HIF-α. More preferably, it does notdirectly affect the activity of any cellular protein other than VDU1. Inparticular, it is preferred that the modulator of VDU1 is not VHL, asVHL directly targets HIF-α for degradation as well as VDU1 (Li et al,2002).

In some embodiments a modulator may be obtainable or obtained by anassay method of the invention, as described above.

An inhibitor or activator of VDU1 may be a natural or synthetic chemicalcompound.

Modulators most suited for therapeutic applications will be smallmolecules e.g. selected from a combinatorial library such as are nowwell known in the art (see e.g. Newton (1997) Expert Opinion TherapeuticPatents, 7(10): 1183-1194). Candidate substances may include smallmolecules such as those of the steroid, benzodiazepine or opiateclasses.

Another class of modulator is polypeptides. An example of an inhibitorpolypeptide is a polypeptide derived from the VHL or VDU1 proteinsequences, which inhibits the interaction between these two proteins.The peptide fragments may be fragments of from 5 to 40 amino acids, forexample from 6 to 10 amino acids from the regions of VHL or VDU1 whichare responsible for the interaction between these proteins.

Other possible modulator polypeptides are anti-VDU1 agonist orantagonist antibodies. Candidate modulator antibodies may becharacterised and their binding regions determined to provide singlechain antibodies and fragments thereof which are responsible formodulating VDU1 activity. Antibodies may be human, or humanised.

A VDU1 antibody is specific, in the sense of being able to distinguishbetween the polypeptide it is able to bind and other polypeptides of thesame species for which it has no or substantially no binding affinity(e.g. a binding affinity of at least about 1000× worse). Specificantibodies bind an epitope on the molecule which is either not presentor is not accessible on other molecules.

Preferred antibodies according to the invention are isolated, in thesense of being free from contaminants such as antibodies able to bindother polypeptides and/or free of serum components. Monoclonalantibodies are preferred for some purposes, though polyclonal antibodiesare within the scope of the present invention.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit) with a polypeptide of the invention. Antibodies maybe obtained from immunised animals using any of a variety of techniquesknown in the art, and screened, preferably using binding of antibody toantigen of interest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al, Nature, 357:80-82,1992).

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, C1 andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

A monoclonal antibody can be subjected to the techniques of recombinantDNA technology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

Preferred activators of VDU1 activity are agents which enhance theexpression of VDU1. Such agents may for example be those which enhancethe production of native VDU1 in the cell, or they may be a nucleic acidwhich encodes VDU1, for example a gene therapy vector designed toexpress VDU1 in target cells.

An inhibitor may be a nucleic acid comprising a sequence correspondingto or complementary to all or part of the sequence of a VDU1 nucleicacid molecule, such that when the modulator is present in a cell VDU1expression is reduced. Such inhibitors may for example be anti-senseRNA, siRNA or a double-stranded RNA which will be processed in the cellto form siRNA, as explained below. Other possible nucleic acidinhibitors include ribozymes which target VDU1 mRNA. These agents may bedirected to VDU mRNA in target cells in the individual, in order toreduce expression of the gene. The nucleic acids may be delivered asnaked nucleic acid or formulations thereof, e.g., liposomal formulationsdesigned to enhance cellular uptake. DNA molecules or gene therapyvectors which express the nucleic acid in the target cell may also beused. Examples of suitable vectors are discussed further below.

RNA interference is a two step process. First, dsRNA is cleaved withinthe cell to yield short interfering RNAs (siRNAs) of about 21-23 ntlength with 5′ terminal phosphate and 3‘short overhangs’ (˜2 nt) ThesiRNAs target the corresponding mRNA sequence specifically fordestruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750,(2001) Thus, the siRNA inhibitor may be a double stranded RNA comprisinga VDU1-encoding sequence, which may for example be a “long” doublestranded RNA (which will be processed to siRNA, e.g., as describedabove). These RNA products may be synthesised in vitro, e.g., byconventional chemical synthesis methods.

RNAi may be also be efficiently induced using chemically synthesizedsiRNA duplexes of the same structure with 3′-overhang ends (Zamore P Det al Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have beenshown to specifically suppress expression of endogenous andheterologeous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)).

Thus a VDU1 inhibitor may also be a siRNA duplex containing between 20and 25 bps, more preferably between 21 and 23 bps, of the VDU1 sequence,e.g. as produced synthetically, optionally in protected form to preventdegradation. Alternatively siRNA may be produced from a vector, in vitro(for recovery and use) or in vivo.

In one embodiment, the vector may comprise a nucleic acid sequencecorresponding to part of the VDU1 sequence in both the sense andantisense orientation, such that when expressed as RNA the sense andantisense sections will associate to form a double stranded RNA. Thismay for example be a long double stranded RNA (e.g., more than 23 nts)which may be processed in the cell to produce siRNAs (see for exampleMyers (2003) Nature Biotechnology 21:324-328). Alternatively, the doublestranded RNA may directly encode the sequences which form the siRNAduplex. In another embodiment, the sense and antisense sequences areprovided on different vectors.

A ribozyme is a catalytic RNA molecule that cleaves other RNA moleculeshaving particular nucleic acid sequences. General methods for theconstruction of ribozymes, including hairpin ribozymes, hammerheadribozymes, RNAse P ribozymes (i.e., ribozymes derived from the naturallyoccurring RNAse P ribozyme from prokaryotes or eukaryotes) are known inthe art. Castanotto et al (1994) Advances in Pharmacology 25: 289-317provides an overview of ribozymes in general, including group Iribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axheadribozymes.

Agents which modulate VDU1 may be administered to a subject in need oftreatment in any suitable form. Usually the agent will be in a form of apharmaceutical composition in which the agent is mixed with apharmaceutically acceptable carrier. The carrier will be adapted to besuitable for the desired route of administration of the agent. The agentmay be administered, for example, orally, topically, subcutaneously orby other routes.

In general, pharmaceutical compositions contemplated for use in thepresent invention can be in the form of a solid, a solution, anemulsion, a dispersion, a micelle, a liposome, and the like, wherein theresulting composition contains one or more of the active compoundscontemplated for use herein, as active ingredients thereof, in admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications. The active ingredients may be compounded,for example, with the usual non-toxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, suppositories, solutions,emulsions, suspensions, and any other form suitable for use. Thecarriers which can be used include glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form.

Pharmaceutical compositions containing the active ingredientscontemplated herein may be in a form suitable for oral use, for example,as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs. Compositions intended for oral use may be prepared according toany method known in the art for the manufacture of pharmaceuticalcompositions.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredients are mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate,kaolin, or the like. They may also be in the form of soft gelatincapsules wherein the active ingredients are mixed with water or an oilmedium, for example, peanut oil, liquid paraffin, or olive oil.

The pharmaceutical compositions may be in the form of a sterileinjectable suspension. This suspension may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides, fatty acids (including oleicacid), naturally occurring vegetable oils like sesame oil, coconut oil,peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyloleate or the like. Buffers, preservatives, antioxidants, and the likecan be incorporated as required.

For example, formulations of compounds for topical administrationinclude transdermal formulations designed to enhance uptake of theactive agent through the skin. Transdermal delivery devices, e.g.patches, are well known in the art and may be used to present atransdermal formulation of the agent.

The amount of agent administered will be dependent upon the nature ofthe agent and its route and dose of administration, whilst also takinginto account the patient and their particular needs.

Gene therapy of somatic cells can be accomplished by using, e.g.,retroviral vectors, other viral vectors, or by non-viral gene transfer(for clarity cf. T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RACDATA MANAGEMENT REPORT, June 1993).

Vector systems suitable for gene-therapy are, for instance, retroviruses(Mulligan, R. C. (1991) in Nobel Symposium 8: Ethiology of human diseaseat the DNA level (Lindsten, J. and Pattersun Editors), pages 143-189,Raven Press), adeno associated virus (McLughlin, J. Virol. 62 (1988),1963), vaccinia virus (Moss et al., Ann. Rev. Immunol. 5 (1987) 305),bovine papilloma virus (Rasmussen et al., Methods Enzymol. 139 (1987)642) or viruses from the group of the herpes viruses such as EpsteinBarr virus (Margolskee et al., Mol. Cell. Biol. 8 (1988) 2937) or Herpessimplex virus.

There are also known non-viral delivery systems. See for example U.S.Pat. No. 6,228,844 (Wolff). For this, usually “nude” nucleic acid,preferably DNA, is used, or nucleic acid together with an auxiliary suchas, e.g., transfer reagents (liposomes, dendromers,polylysine-transferrine-conjugates (Wagner, 1990; Felgner et al., Proc.Natl. Acad. Sci. USA 84 (1987) 7413)).

Gene therapy vectors comprising a sequence encoding VDU1 operably linkedto a promoter functional in the target cells may thus be used tostabilise HIF-α and to increase HIF-mediated responses.

In another embodiment, the gene therapy vector may comprise a sequencewhich corresponds to or is complementary to all or part of the VDU1sequence operably linked to a promotor, which may be used to decreasethe expression of VDU1 and to de-stabilise HIF-α, reducing HIF-mediatedresponses, as discussed above.

Promoters suitable for use in various vertebrate systems are well known.For example, strong promoters include RSV LTR, MPSV LTR, SV40 IEP, andmetallothionein promoter. The CMV IEP may be more preferable for humanuse.

CYLD

As indicated above, individuals with cylindromatosis are those with alesion in the CYLD gene located on chromosome 16q12-13 leading to amutation or lack of expression of the CYLD gene product. Bignell et al,ibid, report the identification of the structure of CYLD and report thatmany affected individuals have mutations located in the 3′ two-thirds ofthe CYLD coding sequence. Other human individuals may have deletions ofthe entire region of the chromosome where the gene is located.

Individuals with cylindromatosis can be administered an effective amountof an NF-κB inhibitor for the treatment of their condition.

By“treatment”, it is meant any degree of alleviation of the diseaseincluding a suppression in the rate of growth of the tumours. This willbe beneficial in lengthening the time before surgical intervention isrequired.

A number of agents which are known to inhibit NF-κB are known in theart. For example, U.S. Pat. No. 5,985,592 discloses that pentoxifyllineor functional derivatives or metabolites thereof can be used for thetreatment of diseases characterised by activation of NF-κB. The phrase“pentoxifylline or functional derivatives/metabolites thereof” refers tothe compound 1-(5-oxohexyl)-3,7-dimethylxanthine (pentoxifylline), andoxidation-, reduction-, substitution- and/or rearrangement-productsthereof, such as, for example, metabolite-1 through metabolite-7 asdescribed by Luke and Rocci in J. Chromatogr.374(1):191-195 (1986)(e.g., 1-(5-hydroxyhexyl)-3,7-dimethyl-xanthine (metabolite-1)), as wellas synthetic variants thereof (e.g., propentofylline).

U.S. Pat. No. 6,090,542 teaches that NF-κB activity may be suppressed bytreating cells with a substance which inhibits the proteolyticdegradation of the alpha subunit of IκB, IκB-α.

Other agents which are known to inhibit NF-κB include aspirin,ibuprofen, sulindac, flurbiprofen and salicylates; and cyclopentenoneprostaglandins (cyPGs) such as A-type and J-type cyPGs, for exampleprostaglandin A1 (PGA1) and cyPG 15-deoxy-delta12-14-PGJ2.

A further class of agents comprises nucleic acids including anti-sensenucleic acids, siRNA and ribozymes. These agents may be directed toNF-κB mRNA in target cells in the individual, in order to reduceexpression of the gene. The nucleic acids may be delivered as naked DNAor formulations thereof, e.g. liposomal formulations designed to enhancecellular uptake. Gene therapy vectors which express the nucleic acids inthe target cells may also be used.

Agents which inhibit NF-κB may be administered to a subject in need oftreatment in any suitable form. Usually the agent will be in a form of apharmaceutical composition in which the agent is mixed with apharmaceutically acceptable carrier. The carrier will be adapted to besuitable for the desired route of administration of the agent. The agentmay be administered, for example, orally, topically, subcutaneously orby other routes.

In general, pharmaceutical compositions contemplated for use in thepresent invention can be in the form of a solid, a solution, anemulsion, a dispersion, a micelle, a liposome, and the like, wherein theresulting composition contains one or more of the active compoundscontemplated for use herein, as active ingredients thereof, in admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications. The active ingredients may be compounded,for example, with the usual non-toxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, suppositories, solutions,emulsions, suspensions, and any other form suitable for use. Thecarriers which can be used include glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form.

Pharmaceutical compositions containing the active ingredientscontemplated herein may be in a form suitable for oral use, for example,as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs. Compositions intended for oral use may be prepared according toany method known in the art for the manufacture of pharmaceuticalcompositions.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredients are mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate,kaolin, or the like. They may also be in the form of soft gelatincapsules wherein the active ingredients are mixed with water or an oilmedium, for example, peanut oil, liquid paraffin, or olive oil.

The pharmaceutical compositions may be in the form of a sterileinjectable suspension. This suspension may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides, fatty acids (including oleicacid), naturally occurring vegetable oils like sesame oil, coconut oil,peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyloleate or the like. Buffers, preservatives, antioxidants, and the likecan be incorporated as required.

For example, formulations of compounds for topical administrationinclude transdermal formulations designed to enhance uptake of theactive agent through the skin.

Transdermal delivery devices, e.g. patches, are well known in the artand may be used to present a transdermal formulation of the agent. Forexample, U.S. Pat. No. 6,368,618 describes a formulation suitable forthe transdermal administration of aspirin comprising aspirin (e.g. in anamount 1% to about 30% w/w) together with at least one alcohol (e.g. inan amount 1% to about 40% w/w) selected from the group consisting ofisopropyl alcohol, ethyl alcohol and propylene glycol; and at least onemelting point depressing agent selected from the group consisting ofthymol, menthol, eucalyptol, eugenol, methyl salicylate, phenylsalicylate, capsaicin, butylated hydroxytoluene, a local anestheticagent and any combination thereof, said melting point depressing agentpresent in the composition in an amount of less than about ¼ (e.g. from1/20 to ¼) of the weight of the aspirin; said composition havingspontaneously equilibrated aqueous and oil phases, wherein the aspirinis in substantially melted form at 25° C., and wherein the concentrationof the aspirin in the oil phase is, by weight, at least about 40% of theweight of the oil phase.

The amount of agent administered will be dependent upon the nature ofthe agent and its route and dose of administration, and taking intoaccount the patient and their particular needs. For example, aspirin andother NSAIDs administered orally can be provided in a unit dosage formof from 100 to 1000 mg, to be taken 1 to 5 times a day. Other routes ofadministration of the same drug may be dosed to an equivalent level.Reference may be made to U.S. Pat. No. 5,985,592 for doses ofpentoxifylline or functional derivatives or metabolites thereof.Prostaglandins may be administered in the range of 0.1 to 100 mg/kg bodyweight per day.

It is possible that CYLD may also be mutated in other cancers, such asbreast, lung, colon and prostate cancers. Thus the agents and theirformulations and routes and doses of delivery referred to herein may beused in the treatment of other cancer conditions associated with amutation in the CYLD gene.

The role of CYLD in suppressing the anti-apoptopic effects of NF-κBprovides the potential to treat diseases associated with cellularproliferation by enhancing the levels of CYLD in a cell in order tosuppress the release of NF-κB from IκB. Such diseases includeinterstitial lung disease, human fibrotic lung disease (e.g., idiopathicpulmonary fibrosis (IPF), adult respiratory distress syndrome (ARDS),tumor stroma in lung disease, systemic sclerosis, Hermansky-Pudlaksyndrome (HPS), coal worker's pneumoconiosis (CWP), chronic pulmonaryhypertension, AIDS associated pulmonary hypertension, and the like),human kidney disease (e.g., nephrotic syndrome, Alport's syndrome,HIV-associated nephropathy, polycystic kidney disease, Fabry's disease,diabetic nephropathy, and the like), glomerular nephritis, nephritisassociated with systemic lupus, liver fibrosis, myocardial fibrosis,pulmonary fibrosis, Grave's ophthalmopathy, drug induced ergotism,cardiovascular disease, cancer, Alzheimer's disease, scarring,scleroderma, glioblastoma in Li-Fraumeni syndrome, sporadicglioblastoma, myeloid leukemia, acute myelogenous leukemia,myelodysplastic syndrome, myeloproliferative syndrome, cancers such asbreast, lung, colon, prostate or gynecological cancer (e.g., ovariancancer, Lynch syndrome, and the like), Kaposi's sarcoma, Hansen'sdisease, inflammatory bowel disease, and the like.

Elevating the levels of CYLD may be achieved by administration of anagent which enhances the production of native CYLD in the cell, or byintroduction of a gene therapy vector designed to express CYLD in targetcells. Gene therapy of somatic cells can be accomplished by using, e.g.,retroviral vectors, other viral vectors, or by non-viral gene transfer(for clarity cf. T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RACDATA MANAGEMENT REPORT, June 1993).

Vector systems suitable for gene therapy are, for instance, retroviruses(Mulligan, R. C. (1991) in Nobel Symposium 8: Ethiology of human diseaseat the DNA level (Lindsten, J. and Pattersun Editors), pages 143-189,Raven Press), adeno associated virus (McLughlin, J. Virol. 62 (1988),1963), vaccinia virus (Moss et al., Ann. Rev. Immunol. 5 (1987) 305),bovine papilloma virus (Rasmussen et al., Methods Enzymol. 139 (1987)642) or viruses from the group of the herpes viruses such as EpsteinBarr virus (Margolskee et al., Mol. Cell. Biol. 8 (1988) 2937) or Herpessimplex virus.

There are also known non-viral delivery systems. See for example U.S.Pat. No. 6,228,844 (Wolff). For this, usually “nude” nucleic acid,preferably DNA, is used, or nucleic acid together with an auxiliary suchas, e.g., transfer reagents (liposomes, dendromers,polylysine-transferrine-conjugates (Wagner, 1990; Felgner et al., Proc.Natl. Acad. Sci. USA 84 (1987) 7413)).

Gene therapy vectors comprising a sequence encoding CYLD (the sequenceof which is available in Bignell et al, ibid, operably linked to apromoter functional in the target cells may thus be used to suppress theanti-apoptopic effects of NF-κB. Promoters suitable for use in variousvertebrate systems are well known. For example, strong promoters includeRSV LTR, MPSV LTR, SV40 IEP, and metallothionein promoter. The CMV IEPmay be more preferable for human use.

Assays according to the invention may be performed in any cell line,preferably a mammalian cell line, more preferably a human cell line, inwhich the NF-κB pathway is active. The cells will either naturallycontain deficient CYLD (e.g. by originating from a subject withcylindromatosis) or may be modified to suppress, either temporally orpermanently, the CYLD gene. Suppression of the activity of CYLD may beachieved by siRNA, as illustrated in the accompanying examples.

In assays of the invention, a cell culture in which CYLD activity issuppressed or missing will be brought into contact with an agent to beassayed. Following incubation of the cells, for example from 1 to 48hours, the activity of NF-κB will be determined.

The amount of an agent which may be added to an assay of the inventionwill normally be determined by trial and error depending upon the typeof compound used. Typically, from about 0.01 to 100 nM concentrations ofagent compounds may be used, for example from 0.1 to 10 nM. Agentcompounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants ormicroorganisms which contain several characterised or uncharacterisedcomponents may also be used.

The activity of NF-κB may be determined by a variety of means. Forexample, the amount of NF-κB protein in a cell may be examined byimmunological techniques, such as western blotting. The amount of NF-κBRNA in the cell may be examined, using for example northern blotting orquantitative PCR. Alternatively, the amount of NF-κB can be examinedusing a reporter gene assay, i.e. by determining the amount ofexpression of a reporter gene (e.g. firefly luciferase, secretedalkaline phosphatase (SEAP) or green fluorescent protein) whose promotercomprises one or more (e.g. two, three or four) tandem copies of the kenhancer element Such constructs are commercially available (e.g. thepNF-κB-Luc vector from BD Biosciences Clontech, Palo Alto, Calif.).

The following examples illustrate the invention.

EXAMPLES

Protein ubiquitination is used primarily to target proteins forproteasome-mediated destruction¹. Protein ubiquitination is a dynamicprocess that involves large families of ubiquitin-conjugating enzymesand ubiquitin ligases that add ubiquitin molecules to substrates and aless-studied family of deubiquitinating enzymes (DUBs) that removeubiquitin from protein substrates. Two classes of DUBs can bedistinguished: ubiquitin C-terminal hydrolases (UCHs) andubiquitin-specific processing proteases (UBPs)^(1,3). The UBP enzymesremove ubiquitin residues from large substrates by cleaving at theC-terminus of the ubiquitin moiety and are candidate antagonists of theubiquitin conjugation/ligation system. A role for DUB genes in cancer issuggested by the fact that this family contains both oncogenes^(6,7) andtumour suppressor genes⁴. In addition, members of the DUB family havebeen described to interact with p53⁸ and BRCA1⁹ and the von HippelLindau (VHL) tumour suppressor gene¹⁰.

Example 1

The strategy we pursued to study the function of the individual-membersof this family of DUB enzymes was to inhibit the expression ofindependent family members through RNA interference and search forphenotypes induced by loss of DUB expression. We first searched severalnucleotide sequence databases for genes with homology to the catalyticdomain of DUBs. A total of 50 genes could be identified harbouring thismotif, including the cylindromatosis tumour suppressor gene (CYLD)⁴ andthe TRE2 oncogene⁶, and DUB no. 33, which corresponds to VDU1.

Next, we retrieved the cDNA sequences corresponding to these potentialDUBs and selected four unique 19-mer sequences from each transcript forcloning into PSUPER, a vector that mediates suppression of geneexpression through the synthesis of short hairpin RNAs having siRNA-likeproperties¹¹. We chose to make four knockdown vectors against each DUBto increase the chance that a significant inhibition of DUB expressionwould be obtained. In total, we made 200 knockdown vectors, which weresubsequently pooled into 50 sets of 4 vectors, where each set of vectorswas designed to target a single DUB transcript.

To ask how effective the set of four knockdown vectors inhibited DUBgene expression, we fused the open reading frame of four of the DUBs toGFP and determined the levels of GFP-DUB fusion protein expression inthe absence and presence of co-expression of the DUB knockdown vectors.293 cells were co-transfected and immunoblotted with a GFP antibody.P21-RFPserved as a transfection control. A significant reduction inprotein levels was induced by all four DUB knockdown vectors, whereascontrol p21-RFP fusion protein was unaffected. We conclude that thisstrategy allows efficient inhibition of DUB expression.

Example 2

The four pSUPER VDU1 knockdown vectors contained the followingsequences: SEQ ID NO:1GATCCCCGAGCCAGTCGGATGTAGATTTCAAGAGAATCTACATCCGACTG GCTCTTTTTGGAAA SEQ IDNO:2 GATCCCCGTAAATTCTGAAGGCGAATTTCAAGAGAATTCGCCTTCAGAAT TTACTTTTTGGAAASEQ ID NO:3 GATCCCCGCCCTCCTAAATCAGGCAATTCAAGAGATTGCCTGATTTAGGAGGGCTTTTTGGAAA SEQ ID NO:4GATCCCCGTTGAGAAATGGAGTGAAGTTCAAGAGACTTCACTCCATTTCT CAACTTTTTGGAAA

Each of the four sequences contains sense and antisense sequence, andform hairpin loops when expressed in cells. These hairpins are convertedby the cell into double-stranded siRNA molecules.

To identify the function individual members of the DUB family, we used ahypoxia inducible reporter (3×RE-luciferase HIFα responsive reporter) tomeasure the effect of the loss of DUB expression. Under normoxicconditions HIF-1α is a protein with a very short half-life due tocontinued VHL mediated ubiquitination. Under hypoxic conditions howeverHIF-1α is rapidly stabilized and functions as a transcriptionalactivator to stimulate transcription of target genes. FIG. 1 shows thatloss of DUB no. 33 in HEP-G2 cells results in decreased activity of thehypoxia inducible reporter under hypoxic conditions (the graph showsinverse values).

As indicated in FIG. 2 VDU-1 knock-down results in a decrease ofreporter activity under both hypoxic and normoxic conditions. SV40Renilla luciferase served as an internal control.

To examine whether loss of VDU-1 not only affects reporter gene activitybut also truly affects the hypoxia response in cells we transfected U2OScells with pSUPER-VDU-1 and pSUPER control vectors and monitored HIF-1αactivation by western blot. As expected 12 hrs treatment of cells withthe hypoxia mimetic desferrioxiamine results in increased HIF-1α,whereas cells transfected with pSUPER-VDU-1 show a marked decrease ofHIF-1α levels under hypoxic conditions (FIG. 3). As HIF-1α is mainlyregulated by ubiquitin induced protein degradation and loss of thedeubiquinating enzyme VDU-1 affects HIF-1α protein levels, it is verylikely that VDU-1 acts on HIF-1α to remove attached ubiquitin chains.This would suggest that full activation of HIF-1α could only be obtainedunder conditions where ubiquitination is inhibited and HIF-1α issubjected to active deubiquitination.

Materials and Methods

Materials, Antibodies, and Plasmids Construction.

To generate DUB knockdown vectors, four annealed sets ofoligonucleotides encoding short hairpin transcripts corresponding to oneDUB enzyme were cloned individually into pSUPER. Bacterial colonies werepooled and used for plasmid preparation. To generate GFP-DUB fusionproteins the corresponding DUB enzymes were PCR amplified using DNA fromhuman cDNA libraries as a template and cloned into pEGFP-N1. The hypoxiainducible reporter plasmid was a 3× hypoxia responsive element linked toluciferase.

Cell Cultures, Transient Transfections and Reporter Assays.

All cells were cultured in Dulbecco's modified Eagle medium (DMEM)supplemented with 10% fetal calf serum. High efficiency electroporationof cells was done as described²⁰. Reporter assays were carried out usingcalcium-phosphate transfection of 0.5 μg 3×RE-luciferase, 1 ngSV40-Renilla and 2.5 μg pSUPER vectors. To mimic hypoxia, cells wereexposed to 12 hrs 1 mM Desferrioxamine (DFO) starting 48 hours aftertransfection.

Immunoblotting

Western blots were performed using whole cell extracts, separated on8-12% SDS-PAGE gels and transferred to polyvinylidine difluoridemembranes (Millipore). Western blots were probed with the indicatedantibodies. Transformed human embryonic kidney cells (293 cells) weretransfected by calcium-phosphate precipitation with the indicatedplasmids, 48 hrs post-transfection cells were lysed in ELB buffer (0.25MNaCL, 0.1% NP-40, 50 mM Hepes pH 7.3) supplemented with “Complete”protease inhibitors (Roche), centrifuged and protein complexes wereimmunoprecipitated with 2 μg of the indicated antibodies conjugated toprotein G sepharose beads. The beads were washed four times with ELBbuffer and protein complexes were eluted by boiling in SDS-sample bufferand resolved on 10% SDS-PAGE.

Example 3

To further study the function of the members of the DUB family, we askedif suppression of any of the DUBs could affect the activity of NF-κB, acancer-relevant transcription factor with marked anti apoptoticactivity¹². We transfected an NF-κB-luciferase reporter gene (3×RE),together with each of the 50 sets of 4 DUB knockdown vectors into humanU2-OS cells and after 48 hours measured the effect of DUB knockdown onTumour Necrosis Factor-α (TNF-α)-activated levels of NF-κB bystimulation overnight with TNF-α (20 ng/ml) and measurement ofluciferase activity. SV40 Renilla luciferase served as an internalcontrol.

The four pSUPER CYLD (DUB36) knockdown vectors contained the followingsequences: SEQ ID NO:5GATCCCCCAGTTATATTCTGTGATGTTTCAAGAGAACATCACAGAATATA ACTGTTTTTGGAAA SEQ IDNO:6 GATCCCCGAGGTGTTGGGGACAAAGGTTCAAGAGACCTTTGTCCCCAACA CCTCTTTTTGGAAASEQ ID NO:7 GATCCCGTGGGCTCATTGGCTGAAGTTCAAGAGACTTCAGCCAATGAGCCCACTTTTTGGAAA SEQ ID NO:8GATCCCCGAGCTACTGAGGACAGAAATTCAAGAGATTTCTGTCCTCAGTA GCTCTTTTTGGAAA

Each of the four sequences contains sense and antisense sequence, andform hairpin loops when expressed in cells. These hairpins are convertedby the cell into double-stranded siRNA molecules.

Only one of the sets of DUB knockdown vectors (#36) significantlyenhanced TNF-α-activation of NF-κB. This effect was specific, asknockdown of DUB#36 did not affect an E2F-luciferase reporter or aHypoxia Induced Factor 1-α (HIF-1α)-responsive promoter (data notshown). Importantly, the DUB#36 set of knockdown vectors targets thecylindromatosis tumour suppressor gene CYLD⁴, a confirmedde-ubiquitinating enzyme¹³, suggesting that CYLD is a regulator ofNF-κB.

To ask if the CYLD knockdown vectors efficiently suppress abundance ofthe CYLD protein, we generated an HA-epitope-tagged CYLD expressionvector and co-transfected this vector with the pSUPER-CYLD knockdownvector, the most active of the four CYLD knockdown vectors in theinitial pool of four CYLD knockdown vectors. U2-OS cells weretransfected with HA-tagged CYLD and pSUPER-CYLD or empty vector. Wholecell extracts were immunoblotted with an HA antibody. GFP served as atransfection control. HA-CYLD protein levels were significantly reducedby pSUPER-CYLD, confirming that CYLD is efficiently targeted forsuppression by the CYLD knockdown vector.

NF-κB is held in an inactive form in the cytoplasm by IκB inhibitorproteins. Signalling through the IκB kinase (IKK) complex, containingthe IκB kinases IKKα and β and the structural component NEMO (or IKKγ),causes phosphorylation and subsequent degradation of IκB, allowingnuclear translocation of NF-κB^(12,14). In principle, the observedeffect of CYLD knockdown on TNF-α stimulation of NF-κB could result froman effect of CYLD on the TNF-α receptor, a more downstream effect on theIKK complex or directly on the IκB/NF-κB complex itself. Since thetumour promoter phorbol 12-myristate 13-acetate (PMA) activates NF-κBdownstream of the TNF-α receptor, we asked if CYLD knockdown alsoaffected PMA-mediated activation of NF-κB. FIG. 4 shows that CYLDknockdown did not enhance basal level of NF-κB activity, but furtherincreased both PMA and TNF-α activated NF-κB levels. This suggests thatCYLD loss affects NF-κB downstream of the TNFα receptor.

Next, we asked if CYLD could physically associate with known members ofthe NF-κB signalling machinery. In this experiment, 293 cells weretransfected as indicated, lysates were prepared 48 hours later and theprotein complexes immunoprecipitated (IP) using Flag antibody. Ips wereimmunoblotted for HA-tagged CYLD and whole cell extracts wereimmunoblotted fro Flag-tagged IκBα, IκKβ and NEMO/IKKγ and for HA-taggedCYLD. CYLD co-immunoprecipitated specifically with NEMO/IKKγ, but notwith IκBa or IκBα. This suggests that CYLD acts on the IκB kinasecomplex through direct association. To address this, we measured IKKβkinase activity following TNF-α stimulation in the presence and absenceof CYLD knockdown, using an in vitro kinase assay. In summary, U2-Scells were co-transfected with Flag-tagged IKKβ and pSUPER-CYLD or emptyvector. Cells were stimulated as indicated, IKKβ was immunoprecipitatedfrom cell lysates and incubated with GST-IκBα (1-72) in the presence of³²P-γATP. Immunoprecipitated IKKβ was visualised by immunoblotting withFlag antibody. In the absence of TNF-α, no IKKβ kinase activity towardsIκBα could be detected. As expected, TNF-α treatment significantlystimulated IKKβ kinase activity. Importantly, this activity was furtherenhanced when cells were co-transfected with CYLD knockdown vector. Noeffects were seen of CYLD knockdown on IKKβ protein levels suggestingthat CYLD does not act to regulate IKKβ abundance.

Consistent with an increase in IKKβ kinase activity by CYLD knockdown,we observed that CYLD knockdown resulted in a more significant reductionin IκBα levels, an endogenous substrate of IKKβ kinase. To test this,U2-OS cells were electroporated with pSUPER-CYLD or empty vector,together with a puromycin resistance marker. Transfected cells wereselected for 48 hours with puromycin (2.0 μg/ml) and stimulated withTNF-α (15 ng/ml). Whole cell extracts were immunoblotted for endogenousIκBα. Together, these data indicate that CYLD acts as an antagonist ofthe IKK complex through direct binding to the non-catalytic NEMO/IKKγcomponent and that reduction of CYLD expression stimulates signallingthrough the IKK complex.

When combined with inhibitors of transcription or translation, TNF-α isa potent inducer of apoptosis in certain cell types.

This pro-apoptotic activity of TNF-α can be inhibited by simultaneousactivation of NF-κB; which activates a number of anti-apoptotic genes¹⁵.Since CYLD knockdown stimulates PMA-induced activation of NF-κB, weasked if CYLD and PMA also collaborate to inhibit TNF-α inducedapoptosis. To address this, we treated Hela cells with TNF-α in thepresence of cycloheximide (CHX) to induce apoptosis both with andwithout pre-treatment with PMA (see methods). 12-hour treatment withTNF-α efficiently induced apoptosis in some 95% of the Hela cells. Asexpected, pre-treatment with PMA resulted in an approximately four-foldincrease of the number of viable cells. Significantly, PMA pre-treatmentin Hela cells that had been transfected with CYLD knockdown vectorresulted in an even larger fraction of surviving cells, suggesting thatloss of the CYLD tumour suppressor gene confers resistance to inductionof apoptosis, most likely through activation of NF-κB. Consistent withthis notion, CYLD knockdown and PMA also collaborated in NF-κBactivation in Hela cells.

NF-κB can be inhibited by a number of pharmacological agents, includingaspirin and prostaglandin A1 (PGA1)^(5,16). Both compounds have beenshown to act on IKKβ, the same kinase that is hyper-activated as aresult of CYLD knockdown. This raises the possibility that the effect ofCYLD knockdown on NF-κB activation can be counteracted by aspirin orPGA1. To address this, we transfected U2-OS cells with theNF-κB-luciferase reporter plasmid and activated NF-κB 48 hours aftertransfection with PMA (200 nM) or PMA and aspirin (10 mM) or PMA andprostaglaridin A1 (8 μM). As was observed before, knockdown of CYLDfurther enhanced PMA-stimulated activation of NF-κB. Strikingly, thiseffect of CYLD knockdown on NF-κB activity could be significantlysuppressed both by aspirin and by PGA1, indicating that these compoundscan compensate for CYLD suppression in this assay.

As was discussed above, it is possible that loss of the CYLD tumoursuppressor gene confers resistance to apoptosis through activation ofNF-κB. If this notion is correct, one would expect that the protectiveeffect of CYLD knockdown on apoptosis can be reversed by the NF-κBinhibitor aspirin. We tested this by treating Hela cells with TNF-α (10ng/ml) and PMA (200 ng/ml) or PMA and aspirin (8 mM) in the presence ofCYLD knockdown. Cycloheximide (10 μg/ml) was used alongside TNF toinduce apoptosis. The combination of CYLD knockdown and PMA treatmentagain conferred significant resistance to TNF-α-induced apoptosis.Significantly, exposure of cells to 10 mM aspirin prior to TNF-αtreatment completely abolished the protective effect of CYLD knockdownon TNF-α-induced apoptosis, indicating that aspirin can also reverse theanti-apoptotic effects of CYLD loss. This result is consistent with thenotion that CYLD knockdown protects from TNF-α-induced apoptosis throughactivation of IKKβ and subsequently of NF-κB.

We describe here the first high-throughput RNA interference screen inmammalian cells to identify novel regulators of NF-κB. We focused onubiquitin-specific processing proteases (UBPs) as these proteins arepotential antagonists of the well-studied ubiquitin conjugating enzymesand ubiquitin ligases. Unexpectedly, we identify the familialcylindromatosis tumour suppressor gene (CYLD) as a novel negativeregulator of NF-κB, thus establishing the first direct link between theNF-κB signalling cascade and a tumour suppressor gene. Our resultsprovide an explanation for the deregulated proliferation of theepidermal appendices in patients with familial mutations in the CYLDgene. It is well-established that NF-κB is required for normal skinproliferation¹⁴. For instance, mice with suppressed NF-κB have defectsin the development of hair follicles and exocrine glands, resulting fromincreased rates of apoptosis¹⁷. Furthermore, female NEMO/IKKγheterozygous mice have severe skin defects, including increasedapoptosis in keratinocytes¹⁸. A similar skin defect is found in thehuman genetic disorder incontinentia pigmenti (IP), which also resultsfrom mutations in the NEMO/IKKγ gene¹⁹. Thus, inhibition of NF-κB in theskin causes an increase in apoptosis. We therefore suggest that thederegulated proliferation in the skin appendices in patients sufferingfrom cylindromatosis results from a perturbation in the balance betweenproliferation and apoptosis in favor of proliferation, resulting from anincrease in active NF-κB. That cylindromas result from a relatively mildperturbation of normal proliferation is also supported by the notionthat most cylindromas have a diploid karyotype and are rarelymetastatic⁴. The observation that the enhanced protection from apoptosisthat results from CYLD suppression can be reversed by simplepharmacological agents like aspirin and prostaglandin A1 suggests astrategy to restore normal growth control in patients suffering fromfamilial cylindromatosis.

Methods.

Materials, Antibodies, and Plasmids Construction.

To generate DUB knockdown vectors, four annealed sets ofoligonucleotides encoding short hairpin transcripts corresponding to oneDUB enzyme were cloned individually into pSUPER. Bacterial colonies werepooled and used for plasmid preparation. To generate GFP-DUB fusionproteins the corresponding DUB enzymes were PCR amplified using DNA fromhuman cDNA libraries as a template and cloned into pEGFP-N1. pNF-κB-Lucvector was obtained from Clontech, SV40-Renilla from Promega. PMA, TNF-αand Prostaglandin A1 and cycloheximide were purchased from Sigma.HA-tagged CYLD was PCR amplified from human cDNA libraries and clonedinto pcDNA 3.1 (−), Flag tagged NEMO was generated by cloning anEcoRI-XbaI NEMO containing fragment into pcDNA-flag. Anti-IκB-α (c-21)and HA tag (Y-11) antibodies were obtained from Santa Cruz, anti-Flag M2from Sigma and anti-GFP rabbit polyclonal serum was kindly provided byJ. Neefjes.

Cell Cultures, Transient Transfections and Reporter Assays.

All cells were cultured in Dulbecco's modified Eagle medium (DMEM)supplemented with 10% fetal calf serum. High efficiency electroporationof cells was done as described²⁰. Reporter assays were carried out usingcalcium-phosphate transfection of 0.5 □g NF-κB-Luc, 1 ng SV40-Renillaand 2.5 μg PSUPER vectors. Forty-eight hours after transfection cellswere stimulated with 200 nM PMA or 20 ng/ml TNF-α and luciferaseactivity was measured 72 hrs post-transfection. Sodium acetylsalicylicacid (10 mM) or Prostaglandin A1 (8 μM) was added to the cells 48 hrsafter transfection, and reporter activity was measured 72 hrs aftertransfection. In Hela cells NF-κB activity was measured 2 hours afterPMA stimulation.

Immunoblotting, Immunoprecipitation and Kinase Assay.

Western blots were performed using whole cell extracts, separated on8-12% SDS-PAGE gels and transferred to polyvinylidine difuoridemembranes (Millipore). Western blots were probed with the indicatedantibodies. Transformed human embryonic kidney cells (293 cells) weretransfected by calcium-phosphate precipitation with the indicatedplasmids, 48 hrs post-transfection cells were lysed in ELB buffer (0.25MNaCL, 0.1% NP-40, 50 mM Hepes pH 7.3) supplemented with “Complete”protease inhibitors (Roche), centrifuged and protein complexes wereimmunoprecipitated with 2 μg of the indicated antibodies conjugated toprotein G sepharose beads. The beads were washed four times with ELBbuffer and protein complexes were eluted by boiling in SDS-sample bufferand resolved on 10% SDS-PAGE. Imunoprecipitation/kinase assays wereperformed essentially as described²¹.

Apoptosis Assays.

Electroporated Hela cells with the indicated plasmids were treated with200 nM PMA for 2-3 hrs 72 hrs post-transfection followed by 12 hrsincubation in medium containing 10 ng/ml TNF-α and 10 μg/mlcycloheximide. Viable cells were quantified using the trypan-blueexclusion method. Alternatively, apoptotic cells were removed by PBSwashing, adherent cells were fixed in 4% paraformaldehyde and stainedusing 0.1% crystal violet (Sigma) and the optical density at 590 nm wasdetermined as described²². To inhibit NF-κB activity medium wassupplemented with 10 mM Sodium acetylsalicylic acid 3.5 hrs before TNF-αaddition. SEQUENCE TABLE SEQ ID NO:1GATCCCCGAGCCAGTCGGATGTAGATTTCAAGAGAATCTACATCCGACTG GCTCTTTTTGGAAA SEQ IDNO:2 GATCCCCGTAAATTCTGAAGGCGAATTTCAAGAGAATTCGCCTTCAGAAT TTACTTTTTGGAAASEQ ID NO:3 GATCCCCGCCCTCCTAAATCAGGCAATTCAAGAGATTGCCTGATTTAGGAGGGCTTTTTGGAAA SEQ ID NO:4GATCCCCGTTGAGAAATGGAGTGAAGTTCAAGAGACTTCACTCCATTTCT CAACTTTTTGGAAA SEQ IDNO:5 GATCCCCCAGTTATATTCTGTGATGTTTCAAGAGAACATCACAGAATATA ACTGTTTTTGGAAASEQ ID NO:6 GATCCCCGAGGTGTTGGGGACAAAGGTTCAAGAGACCTTTGTCCCCAACACCTCTTTTTGGAAA SEQ ID NO:7GATCCCCGTGGGCTCATTGGCTGAAGTTCAAGAGACTTCAGCCAATGAGC CCACTTTTTGGAAA SEQ IDNO:8 GATCCCCGAGCTACTGAGGACAGAAATTCAAGAGATTTCTGTCCTCAGTA GCTCTTTTTGGAAA

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1. An assay method which includes: bringing into contact a putativemodulator and a VDU1 polypeptide; determining whether the putativemodulator binds and/or modulates an activity of VDU1; determining theeffect of the putative modulator on HIF-α stability and/or on theubiquitination state of HIF-α, in a test system comprising HIF-α andVDU1.
 2. An assay method according to claim 1, which includes: bringinginto contact a VDU1 polypeptide with a putative modulator; determiningbinding between the VDU1 polypeptide and the putative modulator;bringing the putative modulator into contact with a test systemcomprising VDU1 and HIF-α; and determining the effect of the putativemodulator on the stability and/or state of ubiquitination of HIF-α. 3.An assay method according to claim 1 which includes: bringing intocontact a VHL polypeptide, a VDU1 polypeptide and a putative modulator;determining whether the putative modulator modulates the interaction ofthe VHL and VDU1 polypeptides; bringing the putative modulator intocontact with a test system comprising VDU1, VHL and HIF-α; determiningthe effect of the putative modulator on the stability and/or state ofubiquitination of HIF-α.
 4. An assay method according to claim 3,wherein the assay method comprises bringing into contact a VHLpolypeptide, a VDU1 polypeptide and a putative modulator compound underconditions where the VHL polypeptide and the VDU1 polypeptide, in theabsence of modulator, are capable of forming a complex.
 5. An assaymethod which includes: bringing a putative modulator into contact withVDU1 and an ubiquitinated VDU1 substrate; determining the ability of theputative modulator to modulate the stabilisation and/or state ofubiquitination of the substrate by VDU1; bringing the putative modulatorinto contact with a test system comprising VDU1 and HIF-α; determiningthe effect of the putative modulator on the stability and/or state ofubiquitination of HIF-α.
 6. An assay method according to claim 1 inwhich the test system further comprises VHL.
 7. An assay methodaccording to claim 1, wherein the test system is a cell.
 8. An assaymethod according to claim 7, wherein the cell is under hypoxicconditions.
 9. An assay method according to claim 7, wherein the cell isunder normoxic conditions.
 10. An assay method according to claim 7,wherein the effect of the putative modulator on HIF-α stability isdetermined by the activity of a HIF-responsive reporter gene.
 11. Anassay method which includes: bringing into contact a putative modulatorwith a test system comprising VDU1 and ubiquitinated HIF-α; determiningthe ability of the putative modulator to modulate the stabilisationand/or state of ubiquitination of HIF-α by VDU1.
 12. An assay methodaccording to claim 11 in which the test system further comprises VHL.13. An assay method according to claim 1, wherein the putative modulatoris brought into contact with the test system under conditions where VDU1is capable of stabilising HIF-α, in the absence of the modulator.
 14. Amodulator of VDU1 for use in a method of medical treatment.
 15. Themodulator according to claim 14 which is an antibody against VDU1. 16.The modulator according to claim 14 which is a nucleic acid comprising asequence encoding VDU1, such that when the modulator is present in acell VDU1 expression is enhanced.
 17. The modulator according to claim14 which is an antisense RNA comprising a sequence which hybridises tothe VDU1 mRNA, a double stranded VDU1 RNA, or a ribozyme which targetsVDU1 RNA, or which is a vector encoding said antisense RNA, doublestranded RNA or ribozyme, such that when the modulator is present in acell VDU1 expression is reduced.
 18. The modulator according to claim14, which is a polypeptide having an amino acid sequence correspondingto a portion of the VHL or VDU1 amino acid sequence, and which bindsspecifically to VHL or VDU1 to prevent VHL and VDU1 from interacting.19. Use of a modulator of VDU1 for the manufacture of a medicament forthe treatment of a condition in which modulation of HIF is oftherapeutic value.
 20. (canceled)
 21. The use according to claim 19which comprises use of an inhibitor of VDU1 for the manufacture of amedicament for the treatment of a condition in which inhibition of HIFactivity is of therapeutic value.
 22. The use according to claim 21,wherein the disease is selected from inflammatory disease, cancer,macular degeneration and diabetic retinopathy, Alzheimer's,atherosclerosis, psoriasis, rheumatoid arthritis and endometriosis. 23.The use according to claim 19, which comprises use of an activator ofVDU1 for the manufacture of a medicament for treatment of a condition inwhich activation of HIF is of therapeutic value.
 24. The use accordingto claim 23, wherein the disease is selected from peripheral andcoronary artery disease and myocardial ischaemia.
 25. A method oftreating a disease in which modulation of HIF is of therapeutic value,the method comprising administering to an individual an effective amountof an agent which modulates the activity of VDU1.
 26. A compositioncomprising a modulator of VDU1 and a pharmaceutically acceptableexcipient.
 27. A composition comprising a modulator according to claim15 and a pharmaceutically acceptable excipient.
 28. A method of treatingan individual with cylindromatosis by administering to the individual aneffective amount of an NF-κB inhibitor.
 29. The method of claim 28wherein said inhibitor is aspirin or prostaglandin A1.
 30. Use of anNF-κB inhibitor for the manufacture of a medicament for the treatment ofcylindromatosis.
 31. Use according to claim 30 wherein said inhibitor isaspirin or prostaglandin A1.
 32. A method of treating a diseaseassociated with activation of NF-κB which in an individual comprisesadministering to an individual an effective amount of an agent whichincreases expression of CYLD.
 33. Use of an agent which increasesexpression of CYLD for the manufacture of a medicament for the treatmentof a disease associated with activation of NF-κB.
 34. An assay methodwhich includes the steps of: providing a cell culture in which CYLDactivity is suppressed or missing; bringing the culture into contactwith an agent to be assayed; and determining the effect of the agent onthe activity of NF-κB.
 35. The method of claim 34 wherein CYLD activityis suppressed using siRNA.
 36. The method of claim 34 wherein the effectof the agent is determined using a reporter gene construct.